Adult stem cells can give rise to entire tissues and this regenerative ability is essential both for repairing tissues after injury, and for maintaining tissues throughout life. Given their important role in normal biology, aging, and disease, there is significant interest in defining the signaling networks that direct stem cell fate. While prior research has identified key regulators of mammary stem/progenitor cell biology, many regulators have yet to be identified, and how these various regulators function within signaling and transcriptional networks to modulate mammary stem/progenitor fate remains poorly understood. Our long-term goal is to define how the internal circuitry of stem/progenitor cells integrates external signals to regulate their fate. We study mammary tissue because of its unique advantages as an experimental model, and because this tissue undergoes multiple cycles of expansion and regression throughout life -- exemplifying the importance of stem cells in adult tissues. This study will contribute essential new knowledge of the signaling networks that regulate stem cell biology by defining a new link between two known regulators of stem cell fate (RUNX1, NOTCH), and by identifying a novel kinase (DDR1) that was previously not known to regulate mammary stem cell fate. The specific objectives of this project are to define how two key regulators, RUNX1 and NOTCH, cooperate to drive the lineage specification of mammary stem/progenitor cells (Aim 1), and to establish how a novel regulator, DDR1, promotes the differentiation of mammary stem and progenitor cells (Aim 2). We will address these objectives with two specific aims: (1) Define the RUNX1- and NOTCH-regulated transcriptional network that drives mammary stem/progenitor differentiation. (2) Define the signaling network activated by DDR1 to regulate stem/progenitor cell differentiation. This study will contribute public health by identifying new genes and signaling networks that regulate stem cell fate in adult tissues, which will facilitate the development of new therapies that harness the power of stem cells for regenerative medicine. This study is experimentally innovative in how it leverages our recently developed hydrogel cultures for growing patient-derived mammary tissues, in synergistic combination with cutting edge genomic approaches ? including CRISPR and single-cell profiling ? to dissect the transcriptional networks and signaling cascades that regulate stem/progenitor fate in human mammary tissue. In summary, these investigations will contribute to our fundamental understanding of how adult stem cell differentiation is genetically controlled, and will yield important knowledge that will be necessary to fully harness the power of adult stem cells for regenerative medicines.